CN109172863A - A kind of method that polycaprolactone-tricalcium phosphate bone tissue engineering scaffold carries out the modification of nanometer decalcifed bone matrix coating - Google Patents
A kind of method that polycaprolactone-tricalcium phosphate bone tissue engineering scaffold carries out the modification of nanometer decalcifed bone matrix coating Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/12—Phosphorus-containing materials, e.g. apatite
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2420/00—Materials or methods for coatings medical devices
- A61L2420/02—Methods for coating medical devices
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Abstract
The invention discloses the methods that a kind of polycaprolactone-tricalcium phosphate bone tissue engineering scaffold carries out the modification of nanometer decalcifed bone matrix coating, the following steps are included: allogeneic nanometer decalcified bone matrix material is mixed with 75% ethyl alcohol, polycaprolactone-tricalcium phosphate bone tissue engineering scaffold is soaked in the nanometer decalcified bone matrix slurry obtained after dilution, it is placed in liquid nitrogen, completes preliminary frost；The frost bracket of acquisition is put into vacuum freeze-drying machine, is lyophilized at least 48 hours, sensitivity is completed, obtains the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold modified through nanometer decalcifed bone matrix coating.The method that the present invention uses low temperature freeze-drying, has been effectively ensured the activity of nano DBM biomaterial；On the basis of retaining script brace aperture, the osteogenic ability of bracket is increased.The present invention provides certain theoretical basis to the development of bone graft substitute and bone impairment renovation material.
Description
Technical field
The present invention relates to bone tissue engineer technical fields, specifically, being related to a kind of polycaprolactone-tricalcium phosphate bone tissue
The method that engineering rack carries out the modification of nanometer decalcifed bone matrix coating.
Background technique
The reparation of bone defect is always the difficult point and hot spot of field of orthopaedics.Currently, treatment bone defect generally use autologous bone,
Allograph bone or tissue engineered bone are filled treatment.Goldstandard of the autologous bone as bone defect healing has ideal induction
Skeletonization, osteoconduction and ostosis characteristic, however its limited source, and there are the complication such as donor site infection, chronic ache.Allosome
Though bone is from a wealth of sources and has good induced osteogenesis and osteoconduction ability, source Control constraints, while having pathogen again
The risks such as propagation, immunological rejection.More scholars concentrate on sight is studied on tissue engineered bone at present.
Bone tissue engineer studies have shown that nature bone is microcosmic on be a kind of multi-level, complicated nanocomposite
([1]Li X,Wang L,Fan Y,Feng Q,Cui FZ,Watari F.Nanostructured scaffolds for
Bone tissue engineering.J Biomed Mater Res is A.2013.101 (8): 2424-35.), inorganic nanometer
Calcium phosphate and hydroxylapatite crystal are deposited between extracellular matrix, and organically with the collagenous fibres of nanostructure and some
The protein molecular of promoting bone growing collectively promote cell growth with Osteoblast Differentiation ([2] Alves CD, Jansen JA,
Leeuwenburgh SC.Synthesis and application of nanostructured calcium phosphate
ceramics for bone regeneration.J Biomed Mater Res B Appl Biomater.2012.100
(8):2316-26.).The easy modification characteristic of organic polymer itself makes it be normally used as the organic moiety of bracket, mainly receives
Rice fiber ([3] Deplaine H, Lebourg M, Ripalda P, et al.Biomimetic hydroxyapatite
coating on pore walls improves osteointegration of poly(L-lactic acid)
scaffolds.J Biomed Mater Res B Appl Biomater.2013.101(1):173-86.).Recent study
Show that proportion structure and the design feature of type i collagen fiber in nature bone of nano fiber scaffold are extremely similar, can effectively facilitate
The adherency of cell is grown, and then promotes regeneration and reconstruction ([4] Gupte MJ, Ma PX.Nanofibrous of bone tissue
scaffolds for dental and craniofacial applications.J Dent Res.2012.91(3):227-
34.).BMSCs is extremely sensitive to nanometer sized materials, and nanostructure can effectively increase the sensing capability of filopodia, promotes thin
Born of the same parents' diffusion and adherency, the 3D microenvironment fast breeding for constituting it in nanofiber, the main reason is that nano material is special
Surface texture can obviously increase specific surface area ([5] Stevens MM, George of protein adsorption and cell adherence
JH.Exploring and engineering the cell surface interface.Science.2005.310
(5751):1135-8.)。
Currently, there are many report about Organic-inorganic composite nano material, it is inorganic with simple hydroxyapatite etc.
Material is compared, and combined support material shows more preferably biocompatibility and osteogenic induction characteristic ([6] Yan LP, Silva-
Correia J,Correia C,et al.Bioactive macro/micro porous silk fibroin/nano-
sized calcium phosphate scaffolds with potential for bone-tissue-engineering
applications.Nanomedicine(Lond).2013.8(3):359-78.[7]Yan LP,Oliveira JM,
Oliveira AL,Caridade SG,Mano JF,Reis RL.Macro/microporous silk fibroin
scaffolds with potential for articular cartilage and meniscus tissue
engineering applications.Acta Biomater.2012.8(1):289-301.[8]Laschke MW,Strohe
A,Menger MD,Alini M,Eglin D.In vitro and in vivo evaluation of a novel
nanosize hydroxyapatite particles/poly(ester-urethane)composite scaffold for
bone tissue engineering.Acta Biomater.2010.6(6):2020-7.[9]Kim H,Che L,Ha Y,
Ryu W.Mechanically-reinforced electrospun composite silk fibroin nanofibers
containing hydroxyapatite nanoparticles.Mater Sci Eng C Mater Biol
Appl.2014.40:324-35.).([10] Chae T, Yang H, Leung V, the Ko F, Troczynski such as Chae
T.Novel biomimetic hydroxyapatite/alginate nanocomposite fibrous scaffolds
For bone tissue regeneration.J Mater Sci Mater Med.2013.24 (8): 1885-94.) by nHAP
Particle is along alginates nanofiber direction uniform deposition, caused by avoiding traditional mechanicalness mixing or method of electrostatic spinning mixing
Inorganic salts aggregation, and effectively promoted osteoblast and adhered in nHAP/ alginates rack surface.Qian etc. ([11] Zhou H,
Touny AH,Bhaduri SB.Fabrication of novel PLA/CDHA bionanocomposite fibers for
tissue engineering applications via electrospinning.J Mater Sci Mater
Med.2011.22 (5): 1183-93.) be prepared for a kind of nHAP/PLGA compound rest (lactic acid polyglycolic acid copolymers with receive
Rice hydroxyapatite), compared with simple nHAP, the biocompatibility of compound rest is remarkably reinforced, and can effectively facilitate MC3T3-
The adherency of E1 cell, proliferation and differentiation.Ganesh N([12]Ganesh N,Ashokan A,Rajeshkannan R,
Chennazhi K,Koyakutty M,Nair SV.Magnetic resonance functional nano-
hydroxyapatite incorporated poly(caprolactone)composite scaffolds for in situ
monitoring of bone tissue regeneration by MRI.Tissue Eng Part A.2014.20(19-
20) a kind of nHAP/PCL compound rest: 2783-94.) is devised, In vitro cell experiment shows compared with simple PCL bracket,
The expression of nHAP/PCL bracket alkaline phosphatase (ALP) increases table of about 43%, the Runx-2 gene on nHAP/PCL bracket
Up to also earlier than simple PCL bracket, this compound rest of explanation with nanostructure has better compared with homogenous material bracket
Osteogenic induction performance.
Although more and more artificial synthesis nano-composite scaffolds materials show good biological tissue's compatibility and
Osteogenic induction performance, but natural bone tissue is handled as Nano grade material and is applied to the rare report of research of rack surface modification
Road.It is suitble to the timbering material of bone tissue regeneration to should also have relatively rich pore structure, pore diameter should at least reach 100 μm, with
Meet absorption and transmitting ([13] Rouwkema J, Rivron NC, the van Blitterswijk of nutriment and oxygen
CA.Vascularization in tissue engineering.Trends Biotechnol.2008.26(8):434-
41.).Research is found to have timbering material optimum bone growth ([14] Murphy of 200-350 μm of pore size of diameter
CM,Haugh MG,O'Brien FJ.The effect of mean pore size on cell attachment,
proliferation and migration in collagen-glycosaminoglycan scaffolds for bone
tissue engineering.Biomaterials.2010.31(3):461-6.).The hole of size unevenness is more advantageous to cell
Proliferation and differentiation ([15] Woodard JR, Hilldore AJ, Lan SK, et al.The mechanical
properties and osteoconductivity of hydroxyapatite bone scaffolds with multi-
scale porosity.Biomaterials.2007.28(1):45-54.).The PCL-TCP bracket holes of preparation experimental group early period
Gap size is 350-450 μm, and porosity 50% meets the best requirement of bone growth.
Ideal bone graft substitute should imitate true bone uptake environment as far as possible certainly.Organizational project is introduced as
The reparation of bone defect provides new thinking.Bracket, seed cell and active growth factor constitute three of bone tissue engineer
Element, wherein especially mostly important with bracket.Ideal bone tissue engineering scaffold should have the following conditions: 1) internal to be interconnected
Pore structure, existing macroporosity (100 μm of aperture >) has microscopic void (aperture is less than 20 μm) again, conducive to the length of tissue
Enter, substance transportation and vascularization；2) biodegradable or absorbable material are made, while it is strong to have both enough biomethanics
Degree and controllable degradation kinetics, can be transmitted to surrounding tissue for weight bearing；3) good interface affinity, conducive to the viscous of cell
Attached, proliferation and differentiation；4) easily prepared at diversified forms and size；5) has the ability of the controlled release active bio factor.
Polycaprolactone (Polycaprolactone, PCL) is a kind of artificial synthesized macromolecular organic compound, is had good
Biocompatibility, biodegradability and plasticity.But itself do not have bioactivity, bad mechanical strength, and speed of degrading
Spend slow, surface is smooth, and hydrophobicity is strong, be not suitable for osteoblast adherency and bone tissue regeneration, be not suitable for bone tissue regeneration, often with
Other one or more biomaterials are used in combination to enhance induced osteogenesis characteristic or biomechanical strength.Wherein, with tricresyl phosphate
Mechanical strength enhancing is more obvious after calcium (Tricalcium phosphate, TCP) is compound, meanwhile, the latter belongs to ceramic calcium class
Inorganic bio, chemical component and crystal structure are similar to natural bone mineral, have good induced osteogenesis, conduction at
Bone characteristic and enough mechanical strengths can promote osteoblast adherency and bone tissue deposition, but brittleness is big, biological absorbable
Property is not good enough.The two can be had complementary advantages by 3D printing technique.Decalcified bone matrix (Demineralized Bone
Matrix, DBM) mainly by 93% collagenous fibres (osteoconduction surface reactive material), 5% soluble protein (induction at
The collaboration such as the BMPs and transforming growth factor of bone, insulin-like growth factor albumen) and 2% remaining mineralized dentin matrix composition,
Its microstructure and natural sclerotin are quite similar, are the commercialization biology materials with good induced osteogenesis, osteoconduction characteristic
Material.Three, which is organically combined, yet there are no document report.
CN201410023751.5 discloses a kind of shaping bone renovating material for Bone Defect Repari, by including following components
Raw material be prepared: α-half-H 2 O calcium sulphate+20-90 parts of hydroxyapatite, 20-40 parts of bioactive minerals powder, autologous bone
Powder particles or 10-80 parts of DBM particle, and the hydrogel for being 1:0.5~1:15 with the total dosage of above-mentioned raw materials.What the present invention obtained
For the plasticity repair materials of Bone Defect Repari, it is suitable for in-situ solidifying, external rapid curing progress Bone Defect Repari after wound injection,
Middle all components have proven to be the good degradation material of biocompatibility.
CN200510027391.7 discloses a kind of preparation side of nanometer decalcified bone matrix material (DBM) for osteanagenesis
Method, including the following steps: 1) use fresh and healthy adult bone, remove soft tissue, obtain various sizes of bone block, flowing water rinses 2-
6hr, dehydrated alcohol are dehydrated 1-3hr, and ether defatting 0.5-2hr, ventilation is dry, are placed in -10 DEG C to -100 DEG C of refrigerator freeze-drying
Afterwards, 0.5-2.5mm particle is ground into sample grinding machine；2) soaked in absolute ethyl alcohol 1-3hr is used, ether impregnates 1-3hr, distilled water flushing
2-5 times, then with salt acid soak 2-8hr, ether impregnates 0.5-2hr, ventilation it is dry decalcified bone matrix DBM, then be placed in -10 DEG C
It is lyophilized to -100 DEG C of refrigerators；3) DBM is ground with grinder to disappear to 1-300nm nano particle, then with gamma-rays or ethylene oxide
Poison is to get nanometer bone-regeneration material.The nano DBM prepared using method of the invention can significantly be changed at following three aspects
The performance of kind DBM: 1) DBM internal particle structure is constructed with nanometer, and monolith intra-graft porosity can be with artificial regulatory, can
So that the bone conduction effect of DBM obtains correct guidance, to meet the needs of all kinds of bone collections.2) hydrophobic after DBM nanometered disposal
Property enhancing, it is more stable after being implanted into vivo, the reduction of DBM antigenicity can be made, osteoinductive activity is more stable.3) application is received
Rice technology building DBM can reduce the consequence because of the decline of biomethanics brought by DBM itself decalcification as far as possible, so that it is more applicable
Bone supporting role when bone grafting fusion.
CN200810202524.3 discloses a kind of homogeneous allogenic bone transplantation substitute material nano DBM, it is characterised in that receives
Grain of rice diameter is 50-100nm, the preparation method is as follows: 1) routinely fresh homogeneous allogenic bone is made using improvement Urist method
DBM bone block；2) DBM bone block is placed in liquid nitrogen frozen pulverizer precomminution, obtains DBM powder；3) nano DBM is prepared: by DBM powder
It is placed in extra-fine grinding pulverizer, appropriate distilled water is added, temperature is maintained at 17 DEG C~25 DEG C, and revolving speed is risen to by 800r/min
Homogenate is carried out high speed centrifugation after being fully ground by 1200r/min, abandons supernatant, obtains nano DBM, dry, packing, sealing, disinfection
?.Using homogeneous allogenic bone as raw material, materials are easy the present invention；Nano DBM can random moulding；Because being nanostructure, make
For bone transplantation substitute material, good biocompatibility, can be used as the exogenous promotion osteanagenesis growth of such as bone morphogenetic protein because
The good carrier of son has good promotion bone regeneration capability.
Summary of the invention
The purpose of the present invention is to provide a kind of polycaprolactone-tricalcium phosphate bone tissue engineering scaffolds to carry out nanometer decalcification bone
The method of matrix granule coating modification, specific method are that one kind passes through desivac for nano DBM grain coating in PCL-TCP bone group
The surface of weaver's engineering support is provided fundamental basis for the design of novel bone impairment renovation material.
To achieve the goals above, The technical solution adopted by the invention is as follows:
The first aspect of the present invention provides a kind of polycaprolactone-tricalcium phosphate bone tissue engineering scaffold progress nanometer decalcification
The method of bone matrix grain coating modification, comprising the following steps:
Allogeneic nanometer decalcified bone matrix (DBM) material is mixed with 75% ethyl alcohol, by polycaprolactone-tricalcium phosphate bone
Tissue engineering bracket (PCL-TCP bone tissue engineering scaffold) is soaked in nanometer decalcified bone matrix (DBM) slurry obtained after dilution,
It is placed in liquid nitrogen, completes preliminary frost；The frost bracket of acquisition is put into vacuum freeze-drying machine, is lyophilized at least 48 hours, is completed
Sensitivity obtains the polycaprolactone-tricalcium phosphate bone tissue engineer branch modified through nanometer decalcifed bone matrix coating
Frame.
The volume ratio of allogeneic nanometer decalcified bone matrix (DBM) material and 75% ethyl alcohol is 1:(5~10), preferably
For 1:5；Concretely 1:5,1:6,1:7,1:8,1:9,1:10.
The polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold) passes through 3D printing
Technology building；3D printing technique is fused glass pellet technology.
The preparation method of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold)
The following steps are included:
By polycaprolactone (Polycaprolactone, PCL) particle and tricalcium phosphate (Tricalcium phosphate,
TCP) powder is according to (3~6): 1 mass ratio premixing, then by the full and uniform mixing of torque rheometer, then by cutting
Grain machine is prepared into the graininess mixing material of suitable 3D printing；
It is first with the matched CAD software design PCL-TCP bone tissue engineering scaffold model fibre diameter of 3D printer
0.5mm, a height of 0.3mm of layer, each layer fiber superposition angle is 0/90 °；Again by above-mentioned graininess mixing material well prepared in advance
It is put into fusion pool, sets fusion pool feeding temperature as 120 DEG C, drop temperature is 110 DEG C, and graininess mixing material is in fusion pool
Inside holding 120 minutes or more, it is ensured that sufficiently melting, the material after thawing are squeezed out by the fine spray head of electric motor boosted micro-syringe formula
Fiber is layering to form polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold).
The preparation method of the allogeneic nanometer decalcified bone matrix material the following steps are included:
The first step prepares nanometer decalcified bone matrix (DBM) bone block: changing using after fresh homogeneous allogenic bone rejecting soft tissue
Good Urist method prepares lumpy nanometer decalcified bone matrix (DBM) bone block；
Second step prepares nanometer decalcified bone matrix (DBM) powder: the lumpy nanometer decalcified bone matrix bone that the first step is obtained
Block is placed in zirconia nanopowder grinding pot, is put into zirconium oxide abrasive ball, is tightened tank mouth, and grinding pot is placed in Full Automatic Liquid chilled nitrogen
Beveller carries out precomminution, obtains nanometer decalcified bone matrix (DBM) powder that partial size is 5-10 μm；
Third step prepares allogeneic nanometer decalcified bone matrix (DBM) material: the nanometer decalcification bone base that second step is obtained
Matter (DBM) powder is placed in zirconia nanopowder grinding pot, and zirconium oxide abrasive ball is added, and it is complete to mill ball that 75% ethyl alcohol is added
Zirconia nanopowder grinding pot is placed in ball milling in full-automatic low temperature high-energy ball milling instrument by covering, and obtaining partial size is the of the same race of 20-50nm
Allosome nanometer decalcified bone matrix (DBM) material.
The improvement Urist method prepare lumpy nanometer decalcified bone matrix (DBM) bone block the following steps are included:
After fresh homogeneous allogenic bone is rejected soft tissue, homogeneous allogenic bone is handled as the bone block of size about 1cm × 1cm,
Allogeneic bone block -80 DEG C of refrigerators are put into after precise weighing to freeze；
Allogeneic bone block after freezing is put into beaker, soaked in absolute ethyl alcohol 2h dehydration；Dehydrated alcohol is outwelled, is added
Ether defatting impregnates 12h；Ether is removed, a large amount of sterile distilled waters rinse 3 times；Configured 0.6mmol/L hydrochloric acid decalcification is added,
72h is impregnated, every 12h replacement hydrochloric acid is primary in soaking process, and is stirred continuously using accurate reinforcement electric mixer, sterile distillation
Water rinses 5 times and soaked overnight；Again with soaked in absolute ethyl alcohol 2h；It goes ethyl alcohol that ether is added and impregnates 1h；Ether is removed, by bone block
Volatilization at ventilation experimental bench is placed on to stay overnight；Packaging seals spare, acquisition lumpy nanometer decalcification bone up for safekeeping after freeze-drying in vacuum freeze-drying machine
Matrix (DBM) bone block.
In the second step, the zirconium oxide abrasive ball for the use of diameter being 1.5cm in zirconia nanopowder grinding pot.
In the second step, 30 time/second of parameter, milling time 5min is arranged in precomminution.
In the third step, the zirconium oxide abrasive ball for the use of diameter being 1mm in zirconia nanopowder grinding pot.
For the third step in full-automatic low temperature high-energy ball milling instrument, temperature is set as 25-35 DEG C, and revolving speed 1500r/min is ground
Consume time 20min, and when temperature is more than 35 DEG C in grinding pot, ball milling instrument will reduce revolving speed to 500 revolutions per seconds, to water cooling unit automatically
When grinding pot temperature is down to 30 DEG C, ball milling instrument will carry out high speed grinding again automatically.
The second aspect of the present invention provides a kind of repairing through nanometer decalcifed bone matrix coating for preparation method preparation
The polycaprolactone of decorations-tricalcium phosphate bone tissue engineering scaffold.
The third aspect of the present invention provides a kind of repairing through nanometer decalcifed bone matrix coating for preparation method preparation
The polycaprolactone of decorations-tricalcium phosphate bone tissue engineering scaffold is preparing answering in bone impairment renovation material or bone graft substitute
With.
Due to the adoption of the above technical scheme, the present invention has the following advantages and beneficial effects:
Polycaprolactone provided by the invention-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcifed bone matrix coating
In the method for modification, use allogeneic nanometer decalcified bone matrix material for homogeneous allogenic bone, materials are easy, and source is wide, can expire
Sufficient clinical demand；Whole process is ground using low temperature-controlled, avoids in process of lapping inside grinding pot heat to collagen in DBM and work
Property protein ingredient destruction, thus preferably retain DBM induced osteogenesis, osteoconduction characteristic；Plasticity is good, can be independent
It uses, can also be used as mixing or coating material uses；Nanoscale DBM biocompatibility is good, is not easy to cause body repulsion
Equal adverse reactions.
Polycaprolactone provided by the invention-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcifed bone matrix coating
In the method for modification, the activity of nano DBM biomaterial has been effectively ensured in the method being lyophilized using low temperature；Retain script bracket
On the basis of hole, the osteogenic ability of bracket is increased.Development of the present invention to bone graft substitute and bone impairment renovation material
Provide certain theoretical basis.
Polycaprolactone provided by the invention-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcifed bone matrix coating
In the method for modification, nano DBM is bioactive materials, can not be added in the 3D printing of pyroprocess directly mixing printing,
Nano DBM is coated on bracket surface layer, is more conducive to adherency, proliferation, migration and the differentiation of the osteoblast of early stage acceleration after surgery,
And the 3D printing that the present invention uses has the advantage of high-precision, the preparation of repeatable and individuation, repairs compared to injection
Multiple material is more flexible.
Detailed description of the invention
Fig. 1 is that polycaprolactone provided in an embodiment of the present invention-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcification bone
Scanning electron microscope in the method for matrix granule coating modification, before polycaprolactone-tricalcium phosphate bone tissue engineering scaffold freeze-drying coating
Figure.
Fig. 2 is that comparative example of the present invention presses polycaprolactone: bone tissue engineering scaffold and the distilled water of tricalcium phosphate 8:2 printing or
The scanning electron microscope (SEM) photograph being lyophilized after the nano DBM particle slurry coating of person's normal saline dilution, it can be seen from the figure that DBM is uneven
Be covered on rack surface and blocking portion brace aperture.
Fig. 3 is that polycaprolactone provided in an embodiment of the present invention-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcification bone
In the method for matrix granule coating modification, polycaprolactone-tricalcium phosphate bone tissue engineering scaffold and the diluted nanometer of 75% ethyl alcohol
The scanning electron microscope (SEM) photograph after coating is lyophilized in DBM particle slurry, it can be seen from the figure that each fiber surface uniform fold nano DBM of bracket,
Bottom is fine and close, is well combined with bracket, and display bracket surface layer is uniformly distributed nano DBM particle, and nano DBM part is reunited, surface
There are nano grooves abundant.
Fig. 4 is polycaprolactone shown in Fig. 3-tricalcium phosphate bone tissue engineering scaffold and the diluted nano DBM of 75% ethyl alcohol
The enlarged drawing of the scanning electron microscope (SEM) photograph after coating is lyophilized in particle slurry.
Fig. 5 is polycaprolactone shown in Fig. 4-tricalcium phosphate bone tissue engineering scaffold and the diluted nano DBM of 75% ethyl alcohol
The enlarged drawing of the scanning electron microscope (SEM) photograph after coating is lyophilized in particle slurry.
Fig. 6 is that polycaprolactone provided in an embodiment of the present invention-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcification bone
In the method for matrix granule coating modification, the scanning electron microscope (SEM) photograph of allogeneic nanometer decalcified bone matrix material.
Fig. 7 is the enlarged drawing of the scanning electron microscope (SEM) photograph of allogeneic nanometer decalcified bone matrix material shown in fig. 6.
Fig. 8 is the enlarged drawing of the scanning electron microscope (SEM) photograph of allogeneic nanometer decalcified bone matrix material shown in Fig. 7.
Specific embodiment
In order to illustrate more clearly of the present invention, below with reference to preferred embodiment, the present invention is described further.Ability
Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, this should not be limited with this
The protection scope of invention.
Main agents and instrument used in the present invention are as follows:
The relative molecular mass 65000 of polycaprolactone is purchased from U.S. Sigma-Aldirch company；Tricalcium phosphate is averaged
Partial size 500nm is purchased from Nanjing Ai Purui nanometers of Co., Ltd, China；Physiological saline is purchased from Cisen Pharmaceutical Co., Ltd., in
State；75% ethyl alcohol is purchased from Chinese medicines group, China；Torque rheometer Rheomix 600Haake Germany；3D printer is Motor
Assisted Microsyringe is purchased from Fu Qifan mechanical & electrical corporation, China；Vacuum freeze-drying machine Christ, Shanghai intelligence Yao's instrument instrument
Table Co., Ltd, China；High-resolution thermal field emission scanning electron microscope Sigma300 is purchased from ZEISS Merlin company, Germany.
Pre-stage test of the present invention successfully constructs PCL-TCP bone tissue engineering scaffold, and prepares nano DBM particle slurry.It closes
Key is how nano DBM particle slurry is effectively adhered PCL-TCP bone tissue engineering scaffold surface.Desivac can be in low temperature
DBM particle slurry is effectively coated on rack surface under environment, and guarantees the activity of DBM biomaterial；Meanwhile preventing to the maximum extent
Only the physics and chemistry of material and biology denaturation, the growth of microorganism and the effect of enzyme can not carry out in drying process, dry in vacuum
Under the conditions of carry out, readily oxidizable substance is protected, therefore is able to maintain original character, and the biomaterial of preservation is lyophilized in Jia Shui
After can dissolve at once, not only have good stability, but also reduce contaminated chance, thus can save longer time.
The present invention is repeatedly attempted nanometer decalcified bone matrix, the mode and PCL-TCP 3D of final application vacuum freeze-drying
Print carriage is mutually compound, to construct a kind of novel organic-inorganic hybrid nanocomposite bracket.By attempting for several times, finally adopt
NDBM/PCL-TCP compound rest is prepared with the mode of vacuum freeze-drying, the reason is that DBM is after nanosizing is ground, liquid nano
Although slurry is sticky, adhesiveness is very poor when in conjunction with bracket, can not bind directly.The present invention is once attempted through rack surface electrostatic
Or the mode of aerial spraying constructs nDBM compound rest, but spraying effect is poor, bracket cannot be combined effectively with nDBM；Then attempt
It is put into natural drying in 37 DEG C of incubator after nDBM slurry after bracket and dilution is impregnated, but nDBM is all deposited on branch after drying
Adhere in the bottom of frame, rack surface and hole without apparent nano slurry.
Using vacuum lyophilization, first PCL-TCP bracket is soaked in nDBM dilution, after -80 DEG C of refrigerator frosts, can be made
NDBM is fixed on state when impregnating with bracket, then so that solid liquid is directly distilled by vacuum freeze-drying machine, fixes nDBM
On scaffold fibers surface.And desivac can prevent the physics and chemistry of material and biology denaturation, micro- life in drying process to the maximum extent
The growth of object and the effect of enzyme can not carry out, and drying carries out under vacuum conditions, and readily oxidizable substance is protected, therefore can be protected
Original character is held, the biomaterial that preservation is lyophilized can dissolve at once after adding the water, not only have good stability, but also reduce
Contaminated chance, thus longer time ([15] Woodard JR, Hilldore AJ, Lan SK, et al.The can be saved
mechanical properties and osteoconductivity of hydroxyapatite bone scaffolds
with multi-scale porosity.Biomaterials.2007.28(1):45-54.)。
Nano material disperses to need suitable dispersing agent in the liquid phase, and the dispersion (dispersing agent) of nanometer decalcified bone matrix has no
Relevant report, for the destruction for avoiding nDBM bioactivity, dilute the solution selected be sterile distilled water, physiological saline and allograph bone
Ethyl alcohol used when decalcification.In dilution, distilled water and physiological saline are starched with nDBM after mixing, and it is existing that agglomeration solidification occurs in nDBM
As showing that nDBM is not readily dissolved in distilled water and physiological saline；After 75% ethyl alcohol of 1:5 is added into nDBM slurry, nDBM dispersion is equal
Even, no solidification and deposited phenomenon occur；And when nDBM and ethyl alcohol diluted concentration are 1:1,1:2, nDBM disperses in rack surface
Unevenly, Partial Blocking brace aperture, when diluted concentration is 1:10, nDBM concentration is too low, and portion support fiber surface is without nDBM
Attachment, this is also the reason of ethyl alcohol of final choice 1:5 is as nDBM dilution.
Embodiment 1
A kind of polycaprolactone-tricalcium phosphate bone tissue engineering scaffold carries out the side of nanometer decalcifed bone matrix coating modification
Method, comprising the following steps:
One, allogeneic nanometer decalcified bone matrix (DBM) material is mixed with 75% ethyl alcohol, dilutes nano slurry
By allogeneic nanometer decalcified bone matrix (DBM) material (embodiment 3 is prepared) and 75% ethyl alcohol according to volume
Than the ratio mixing for 1:5, fulling shake is uniformly distributed nano DBM particle in ethanol.
Mixing condition is as follows: 20-25 DEG C at room temperature, instrument: desk-top constant-temperature table NHWY-100B, temperature control range are 5~60
DEG C (use 30 DEG C of constant temperature), 40~300rmp of revolving speed, amplitude range Ф 26mm, Changzhou Nuo Ji Instrument Ltd., time is
5min。
Two, preliminary frost
It is received what PCL-TCP bone tissue engineering scaffold immersion (20-25 DEG C at room temperature, impregnate 2 minutes) obtained after dilution
Rice decalcified bone matrix (DBM) is placed in 20ml plastic test tube, fullys shake again, be then at once placed in plastic test tube in starching
In liquid nitrogen, completing preliminary frost, (preliminary the used instrument of frost commonly holds liquid nitrogen examination ware and common plastics examination without particular/special requirement
Guan Junke).The polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold) passes through 3D printing
Technology building；3D printing technique is fused glass pellet technology.
The preparation method of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold)
The following steps are included:
By polycaprolactone (Polycaprolactone, PCL) particle and tricalcium phosphate (Tricalcium phosphate,
TCP) powder is pre-mixed according to the mass ratio of 8:2, then by the full and uniform mixing of torque rheometer, then passes through pelletizing mechanism
The standby graininess mixing material at suitable 3D printing.
First with the matched CAD software of 3D printer, (HTS Slice Software is purchased from Fu Qifan company.) design PCL-TCP
Bone tissue engineering scaffold model fibre diameter is 0.5mm, and a height of 0.3mm of layer, each layer fiber superposition angle is 0/90 °；It again will be upper
It states graininess mixing material well prepared in advance to be put into fusion pool, sets fusion pool feeding temperature as 120 DEG C, drop temperature is
110 DEG C, graininess mixing material was at fusion pool inside holding 120 minutes or more, it is ensured that sufficiently melting, the material after thawing pass through electricity
Machine aided pushes away the fine spray head of micro-syringe formula and squeezes out fiber, is layering to form polycaprolactone-tricalcium phosphate bone tissue engineering scaffold
(PCL-TCP bone tissue engineering scaffold).
Three, coating modification is completed in freeze-drying
Bracket after preliminary frost is put into vacuum freeze-drying machine, 48 hours completion sensitivities are lyophilized, are passed through
Polycaprolactone-tricalcium phosphate bone tissue engineering scaffold of nanometer decalcifed bone matrix coating modification.
Vacuum freeze-drying machine: Labconco company of the U.S., FreeZone freeze dryer, -84 DEG C of condenser temperature, vacuum degree: <
1.5mbar, freeze-drying time 48 hours.
Four, scanning electron microscopic observation
Polycaprolactone-tricalcium phosphate the bone tissue modified through nanometer decalcifed bone matrix coating that will be obtained in step 3
After engineering rack metal spraying sample preparation, the pattern variation of rack surface is observed under scanning electron microscope.As shown in Fig. 1,3~5, Fig. 1 is this
The polycaprolactone that inventive embodiments provide-tricalcium phosphate bone tissue engineering scaffold carries out the modification of nanometer decalcifed bone matrix coating
Method in, the scanning electron microscope (SEM) photograph before polycaprolactone-tricalcium phosphate bone tissue engineering scaffold freeze-drying coating.Fig. 3 is that the present invention is real
Apply the method that polycaprolactone-tricalcium phosphate bone tissue engineering scaffold of example offer carries out the modification of nanometer decalcifed bone matrix coating
In, polycaprolactone-tricalcium phosphate bone tissue engineering scaffold and sweeping after the diluted nano DBM particle slurry freeze-drying coating of 75% ethyl alcohol
Electron microscope is retouched, it can be seen from the figure that each fiber surface uniform fold nano DBM of bracket, bottom is fine and close, good in conjunction with bracket
Good, display bracket surface layer is uniformly distributed nano DBM particle, and nano DBM part is reunited, and there are nano grooves abundant on surface.Fig. 4 is
Coating is lyophilized in polycaprolactone shown in Fig. 3-tricalcium phosphate bone tissue engineering scaffold and the diluted nano DBM particle slurry of 75% ethyl alcohol
The enlarged drawing of scanning electron microscope (SEM) photograph afterwards.Fig. 5 is polycaprolactone shown in Fig. 4-tricalcium phosphate bone tissue engineering scaffold and 75% second
The enlarged drawing of scanning electron microscope (SEM) photograph after the diluted nano DBM particle slurry freeze-drying coating of alcohol.From Fig. 1,3,4,5 as can be seen that bracket
Each fiber surface uniform fold nano DBM, bottom is fine and close, is well combined with bracket, shows the equally distributed nanometer in bracket surface layer
DBM particle, nano DBM part are reunited, and surface has nano grooves abundant (Fig. 3,4,5).
As a result illustrate: being repaired with the nano DBM grain coating that method of the invention completes PCL-TCP bone tissue engineering scaffold
Decorations, nano DBM particle are uniformly distributed.
Embodiment 2
A kind of polycaprolactone-tricalcium phosphate bone tissue engineering scaffold carries out the side of nanometer decalcifed bone matrix coating modification
Method, comprising the following steps:
One, allogeneic nanometer decalcified bone matrix (DBM) material is mixed with 75% ethyl alcohol, dilutes nano slurry
By allogeneic nanometer decalcified bone matrix (DBM) material (embodiment 3 is prepared) and 75% ethyl alcohol according to volume
Than the ratio mixing for 1:10, fulling shake is uniformly distributed nano DBM particle in ethanol.
Remaining coating step is the same as embodiment 1.
Embodiment 3
The preparation method of adult allogeneic nanometer decalcified bone matrix material the following steps are included:
One, improvement Urist method is used to prepare lumpy nanometer decalcification bone after fresh adult homogeneous allogenic bone being rejected soft tissue
Matrix (DBM) bone block
(1) bone tissue source
Homogeneous allogenic bone used in bone tissue decalcification, derives from attached Long March hospital of army medical university of naval 2015 to 2016
Femoral head, the tibial plateau, condyle of femur part cut off in year joint surgery row hip joint, knee prosthesis patient's art, through Shanghai
The examination & approval of Long March hospital Medical Ethics Committee are agreed to be used for clinical research.Selected donor be all satisfied American tissue library association as
Lower regulation: without acute and chronic infectious diseases history；The contact of nonhazardous substance and history of drug abuse；Without diseases such as tumour, sexually transmitted disease, hepatitis
History；No radiotherapy and chemotherapy medicine or long-term, high-dose hormone application history；Preoperative three big routine test checks normal.Allogeneic is collected altogether
Bone 1400.9g.
(2) homogeneous allogenic bone is handled
Marrow in the soft tissue, periosteum and pulp cavity of homogeneous allogenic bone attachment is rejected, periarticular bone tissue needs as far as possible
Reject articular surface cartilage, sterile distilled water rinse after, with the tools such as rongeur by homogeneous allogenic bone handle for size about 1cm ×
Allogeneic bone block is put into -80 DEG C of refrigerators and frozen by the bone block of 1cm after the weighing of precision electronic balance instrument.
(3) lumpy nanometer decalcified bone matrix (DBM) bone block is prepared using improvement Urist method
Allogeneic bone block after step (2) is frozen is put into the beaker of 2000ml, soaked in absolute ethyl alcohol 2h dehydration；?
Fall dehydrated alcohol, ether defatting is added, impregnates 12h；Ether is removed, a large amount of sterile distilled waters rinse 3 times；It is added configured
72h is impregnated in 0.6mmol/L hydrochloric acid decalcification, and every 12h replacement hydrochloric acid is primary in soaking process, and uses accurate reinforcement electric stirring
Device is stirred continuously, and guarantees that bone block and hydrochloric acid come into full contact with simultaneously decalcification；Sterile distilled water rinses 5 times and soaked overnight, to guarantee to fill
Divide and removes demineralizing acid；Again with soaked in absolute ethyl alcohol 2h；It goes ethyl alcohol that ether is added and impregnates 1h；Ether is removed, bone block is placed on logical
It volatilizees overnight at wind experimental bench；Packaging seals spare, acquisition lumpy nanometer decalcified bone matrix (DBM) up for safekeeping after freeze-drying in vacuum freeze-drying machine
Bone block 257g.
Two, nanometer decalcified bone matrix (DBM) powder is prepared
Appropriate freeze-drying lumpy nanometer decalcified bone matrix (DBM) bone block is taken to be fitted into the zirconium oxide abrasive tank that capacity is 30ml,
It is put into the zirconium oxide abrasive ball that diameter is 1.5cm, after tightening tank mouth, grinding pot merging CryoMill Full Automatic Liquid chilled nitrogen is ground
It grinds instrument (Lai Chi company, Germany) and carries out precomminution, 30 time/second of parameter is set, and milling time 5min, obtaining partial size is 5-10 μm
Nanometer decalcified bone matrix (DBM) powder.In entire process of lapping, the addition and supplement of liquid nitrogen, the pre-cooling of instrument, certainly by program
Dynamic control.
Three, allogeneic nanometer decalcified bone matrix (DBM) material is prepared:
It is to be added in the zirconia nanopowder grinding pot of 50ml by above-mentioned nanometer decalcified bone matrix (DBM) powder merging capacity
Diameter is the zirconium oxide abrasive ball of 1mm, after mixing with DBM, 75% ethyl alcohol to mill ball is added and is completely covered.Then by nanometer
Grinding pot is placed in the full-automatic low temperature high-energy ball milling instrument of E-Max (E-Max, Lai Chi company, Germany), setup parameter are as follows: 1500r/
S, time 20min, set temperature threshold value are 25 DEG C -35 DEG C, and when temperature is more than 35 DEG C in grinding pot, ball milling instrument will reduce automatically
Revolving speed is to 500 revolutions per seconds, and when grinding pot temperature is down to 30 DEG C by water cooling unit, ball milling instrument will carry out high speed grinding again automatically；?
Allogeneic nanometer decalcified bone matrix (DBM) material for being 20-50nm to partial size, suction pipe are put into 20ml plastic test tube after being sucked out
It is interior.
Scanning electron microscopic observation
After allogeneic nanometer decalcified bone matrix (DBM) the material metal spraying sample preparation obtained in step 3, in scanning electron microscope
The pattern on lower observation surface.As can be seen from figures 6 to 8, Fig. 6 is polycaprolactone provided in an embodiment of the present invention-tricalcium phosphate bone tissue
Engineering rack carries out in the method for nanometer decalcifed bone matrix coating modification, and allogeneic nanometer decalcified bone matrix material is swept
Electron microscope is retouched, Fig. 7 is the enlarged drawing of the scanning electron microscope (SEM) photograph of allogeneic nanometer decalcified bone matrix material shown in fig. 6, and Fig. 8 is figure
The enlarged drawing of the scanning electron microscope (SEM) photograph of allogeneic nanometer decalcified bone matrix material shown in 7；As can be seen that DBM from Fig. 6~8
Bottom is fine and close, is connected with each other in threadiness, fiber thickness etc., diameter 40-80nm, and surface is full of irregular nano particle,
Granular size is uneven, and diameter 20nm-50nm, nano particle is mutually reunited, and gather Nano grade groove on surface, nanostructure
Fiber is connected with each other, and inside forms more micron level pore structure, and pore size is different, communicates with one another.
As a result illustrate: with the DBM of improvement Urist method preparation, after low-temperature physics is ground, obtained nano DBM is micro-
It sees structure and meets nano meter biomaterial scope.
Embodiment 4
The preparation method of rabbit allogeneic nanometer decalcified bone matrix material the following steps are included:
Used fresh bone 100g comes from new zealand rabbit (offer of army medical university of naval animal experimental center), remaining is of the same race
Allosome nanometer decalcified bone matrix material preparation process is the same as embodiment 3.
Embodiment 5
A kind of polycaprolactone-tricalcium phosphate bone tissue engineering scaffold carries out the side of nanometer decalcifed bone matrix coating modification
Method, comprising the following steps:
One, allogeneic nanometer decalcified bone matrix (DBM) material is mixed with 75% ethyl alcohol, dilutes nano slurry
By allogeneic nanometer decalcified bone matrix (DBM) material (embodiment 3 is prepared) and 75% ethyl alcohol according to volume
Than the ratio mixing for 1:7.5, fulling shake is uniformly distributed nano DBM particle in ethanol.
Remaining coating step is the same as embodiment 1.
Comparative example 1
One, allogeneic nanometer decalcified bone matrix (DBM) material is mixed with distilled water or physiological saline, dilutes nano slurry
By allogeneic nanometer decalcified bone matrix (DBM) material (embodiment 3 is prepared) and distilled water or physiology salt
Water is mixed according to the ratio that volume ratio is 1:5, and fulling shake makes nano DBM particle be evenly distributed on distilled water or physiological saline
In.
Two, preliminary frost
PCL-TCP bone tissue engineering scaffold is soaked in nanometer decalcified bone matrix (DBM) slurry obtained after dilution, is placed in
It in 20ml plastic test tube, fullys shake, plastic test tube is placed in liquid nitrogen at once again then, complete preliminary frost.It is described poly-
Caprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold) is constructed by 3D printing technique；3D printing
Technology is fused glass pellet technology.
The preparation method of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold)
The following steps are included:
By polycaprolactone (Polycaprolactone, PCL) particle and tricalcium phosphate (Tricalcium phosphate,
TCP) powder is pre-mixed according to the mass ratio of 8:2, then by the full and uniform mixing of torque rheometer, then passes through pelletizing mechanism
The standby graininess mixing material at suitable 3D printing.
First with the matched CAD software of 3D printer, (HTS Slice Software is purchased from Fu Qifan company.) design PCL-TCP
Bone tissue engineering scaffold model fibre diameter is 0.5mm, and a height of 0.3mm of layer, each layer fiber superposition angle is 0/90 °；It again will be upper
It states graininess mixing material well prepared in advance to be put into fusion pool, sets fusion pool feeding temperature as 120 DEG C, drop temperature is
110 DEG C, graininess mixing material was at fusion pool inside holding 120 minutes or more, it is ensured that sufficiently melting, the material after thawing pass through electricity
Machine aided pushes away the fine spray head of micro-syringe formula and squeezes out fiber, is layering to form polycaprolactone-tricalcium phosphate bone tissue engineering scaffold
(PCL-TCP bone tissue engineering scaffold).
Three, coating modification is completed in freeze-drying
Bracket after preliminary frost is put into vacuum freeze-drying machine, 48 hours completion sensitivities are lyophilized.Scanning electricity
Sem observation as shown in Fig. 2, Fig. 2 be comparative example of the present invention press polycaprolactone: tricalcium phosphate 8:2 printing bone tissue engineering scaffold and
The scanning electron microscope (SEM) photograph being lyophilized after the nano DBM particle slurry coating of distilled water or normal saline dilution, it can be seen from the figure that
DBM is non-uniform to be covered on rack surface and blocking portion brace aperture.Bracket passes through and distilled water or normal saline dilution
Nano DBM particle slurry impregnate after, nano DBM is non-uniform to be covered on rack surface and blocking portion brace aperture, nanoscale
Other DBM particle and groove are unobvious, are in dense thin sheet, partially in loose netted.
Comparative example 2
The present invention once attempted to construct nDBM compound rest by way of rack surface electrostatic or aerial spraying, but sprayed effect
Fruit is poor, and bracket cannot be combined effectively with nDBM, macroscopic result: nano slurry can not adhere to or adhere in rack surface.
One, the step of aerial spraying: starching nDBM at 20-25 DEG C of room temperature and dilute mixing with 75% ethyl alcohol, is placed in Finex
In air gun supplying bottle, muzzle alignment PCL-TCP rack surface is sprayed, and visually observe: nDBM can not effectively and bracket
Adherency, and nDBM easily blocks muzzle in spraying process, adheres in rack surface and hole without apparent nano slurry.
Two, the step of electrostatic spraying: starching nDBM at 20-25 DEG C of room temperature and dilute mixing with 75% ethyl alcohol, is placed in Graco
In Pro Xp air auxiliary electrostatic spray gun supplying bottle, muzzle alignment PCL-TCP rack surface is sprayed, and is visually observed: nDBM
It can not effectively be adhered to bracket, and nDBM easily blocks muzzle in spraying process, nothing is significantly received in rack surface and hole
Rice & peanut milk attachment.
Comparative example 3
NDBM after polycaprolactone-tricalcium phosphate bone tissue engineering scaffold and dilution is starched and is impregnated, 37 DEG C of incubator is put into
Interior natural drying, but nDBM is all deposited on the bottom of bracket after drying, and rack surface and hole are interior attached without apparent nano slurry
?.
Specific steps: by the nDBM after polycaprolactone-tricalcium phosphate bone tissue engineering scaffold and dilution at 20-25 DEG C of room temperature
Slurry impregnates, and is put into 37 DEG C of incubator, takes out after drying 20min, and visually observe: nDBM is all deposited on the bottom of bracket, branch
Adhere in frame surface and hole without apparent nano slurry, nano slurry can not adhere to or adhere in rack surface.
Comparative example 4
Prepare allogeneic nanometer decalcified bone matrix (DBM) material:
It is to be added in the zirconia nanopowder grinding pot of 50ml by above-mentioned nanometer decalcified bone matrix (DBM) powder merging capacity
Diameter is the zirconium oxide abrasive ball of 1mm, after mixing with DBM, 75% ethyl alcohol to mill ball is added and is completely covered.Then by nanometer
Grinding pot is placed in the full-automatic low temperature high-energy ball milling instrument of E-Max (E-Max, Lai Chi company, Germany), setup parameter are as follows: 1500r/
S, time 20min, set temperature threshold value are 25 DEG C -35 DEG C, and when temperature is more than 35 DEG C in grinding pot, ball milling instrument will reduce automatically
Revolving speed is to 500 revolutions per seconds, and when grinding pot temperature is down to 30 DEG C by water cooling unit, ball milling instrument will carry out high speed grinding again automatically；?
Allogeneic nanometer decalcified bone matrix (DBM) material for being 20-50nm to partial size, suction pipe are put into 20ml plastic test tube after being sucked out
It is interior.
It is ground in allogeneic nanometer decalcified bone matrix (DBM) material preparation process (i.e. process of lapping) using ethyl alcohol,
Principle is consistent with dilution principle, needs to guarantee that particle effectively disperses in liquid in process of lapping, just can guarantee that DBM is fully ground
To Nano grade, i.e., ground using ethyl alcohol as solvent, grinding effect is good, DBM effectively can be ground to nanoscale；And it uses
Water is ground as solvent, and DBM dispersion is uneven, and particle is still very coarse after twenty minutes for grinding, can not effectively be ground into nano DBM
Slurry is also not improved even if extending milling time.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent defines.
Claims (10)
1. the side that a kind of polycaprolactone-tricalcium phosphate bone tissue engineering scaffold carries out the modification of nanometer decalcifed bone matrix coating
Method, it is characterised in that: the following steps are included:
Allogeneic nanometer decalcified bone matrix material is mixed with 75% ethyl alcohol, by polycaprolactone-tricalcium phosphate bone tissue engineer
Bracket is soaked in the nanometer decalcified bone matrix slurry obtained after dilution, is placed in liquid nitrogen, and preliminary frost is completed；By the frost of acquisition
Bracket is put into vacuum freeze-drying machine, is lyophilized at least 48 hours, and sensitivity is completed, and is obtained through nanometer decalcifed bone matrix
Polycaprolactone-tricalcium phosphate bone tissue engineering scaffold of coating modification.
2. polycaprolactone according to claim 1-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcified bone matrix
The method of grain coating modification, it is characterised in that: the volume ratio of the allogeneic nanometer decalcified bone matrix material and 75% ethyl alcohol
For 1:(5~10), preferably 1:5.
3. polycaprolactone according to claim 2-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcified bone matrix
The method of grain coating modification, it is characterised in that: the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold passes through 3D printing technique
Building；3D printing technique is fused glass pellet technology；
Preferably, the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold preparation method the following steps are included:
By polycaprolactone particle and tricalcium phosphate powder according to (3~6): then 1 mass ratio premixing passes through torque rheology
The full and uniform mixing of instrument, then it is prepared by pelleter the graininess mixing material of suitable 3D printing；
Polycaprolactone-tricalcium phosphate bone tissue engineering scaffold model fibre is designed first with the matched CAD software of 3D printer
Diameter is 0.5mm, and a height of 0.3mm of layer, each layer fiber superposition angle is 0/90 °；Above-mentioned graininess well prepared in advance is mixed again
Condensation material is put into fusion pool, sets fusion pool feeding temperature as 120 DEG C, drop temperature is 110 DEG C, and graininess mixing material exists
Fusion pool inside holding 120 minutes or more, it is ensured that sufficiently melting, the material after thawing are finely sprayed by electric motor boosted micro-syringe formula
Head squeezes out fiber, is layering to form polycaprolactone-tricalcium phosphate bone tissue engineering scaffold.
4. polycaprolactone according to claim 3-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcified bone matrix
The method of grain coating modification, it is characterised in that: the preparation method of the allogeneic nanometer decalcified bone matrix material includes following
Step:
The first step prepares nanometer decalcified bone matrix bone block: using improvement Urist method after fresh homogeneous allogenic bone is rejected soft tissue
Prepare lumpy nanometer decalcified bone matrix bone block；
Second step prepares nanometer decalcified bone matrix powder: the lumpy nanometer decalcified bone matrix bone block that the first step is obtained is placed in oxygen
Change the nano-milled tank of zirconium in, be put into zirconium oxide abrasive ball, tighten tank mouth, by grinding pot be placed in Full Automatic Liquid chilled nitrogen beveller into
Row precomminution obtains the nanometer decalcified bone matrix powder that partial size is 5-10 μm；
Third step prepares allogeneic nanometer decalcified bone matrix material: the nanometer decalcified bone matrix powder that second step obtains is set
Enter in zirconia nanopowder grinding pot, zirconium oxide abrasive ball is added, 75% ethyl alcohol to mill ball is added and is completely covered, by zirconium oxide
Nano-milled tank is placed in ball milling in full-automatic low temperature high-energy ball milling instrument, obtains the allogeneic nanometer decalcification that partial size is 20-50nm
Extracellular matrix material.
5. polycaprolactone according to claim 4-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcified bone matrix
The method of grain coating modification, it is characterised in that: it includes following that the improvement Urist method, which prepares lumpy nanometer decalcified bone matrix bone block,
Step:
After fresh homogeneous allogenic bone is rejected soft tissue, homogeneous allogenic bone is handled as the bone block of size about 1cm × 1cm, precision
Allogeneic bone block -80 DEG C of refrigerators are put into after weighing to freeze；
Allogeneic bone block after freezing is put into beaker, soaked in absolute ethyl alcohol 2h dehydration；Dehydrated alcohol is outwelled, ether is added
12h is impregnated in degreasing；Ether is removed, a large amount of sterile distilled waters rinse 3 times；Configured 0.6mmol/L hydrochloric acid decalcification is added, impregnates
72h, every 12h replacement hydrochloric acid is primary in soaking process, and is stirred continuously using accurate reinforcement electric mixer, sterile distilled water punching
Wash 5 times and soaked overnight；Again with soaked in absolute ethyl alcohol 2h；It goes ethyl alcohol that ether is added and impregnates 1h；Ether is removed, bone block is placed
Volatilization is stayed overnight at ventilation experimental bench；Packaging seals spare, acquisition lumpy nanometer decalcified bone matrix up for safekeeping after freeze-drying in vacuum freeze-drying machine
Bone block.
6. polycaprolactone according to claim 4-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcified bone matrix
The method of grain coating modification, it is characterised in that: it is 1.5cm's that diameter is used in the second step, in zirconia nanopowder grinding pot
Zirconium oxide abrasive ball；
In the second step, 30 time/second of parameter, milling time 5min is arranged in precomminution.
7. polycaprolactone according to claim 4-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcified bone matrix
The method of grain coating modification, it is characterised in that: in the third step, the oxygen for the use of diameter being 1mm in zirconia nanopowder grinding pot
Change zirconium mill ball.
8. polycaprolactone according to claim 4-tricalcium phosphate bone tissue engineering scaffold carries out nanometer decalcified bone matrix
The method of grain coating modification, it is characterised in that: for the third step in full-automatic low temperature high-energy ball milling instrument, temperature is set as 25-
35 DEG C, revolving speed 1500r/min, milling time 20min, when temperature is more than 35 DEG C in grinding pot, ball milling instrument will reduce automatically to be turned
Speed is to 500 revolutions per seconds, and when grinding pot temperature is down to 30 DEG C by water cooling unit, ball milling instrument will carry out high speed grinding again automatically.
9. a kind of any one of claim 1 to the 8 preparation method preparation through the modification of nanometer decalcifed bone matrix coating
Polycaprolactone-tricalcium phosphate bone tissue engineering scaffold.
10. a kind of any one of claim 1 to the 8 preparation method preparation through the modification of nanometer decalcifed bone matrix coating
Polycaprolactone-tricalcium phosphate bone tissue engineering scaffold is preparing the application in bone impairment renovation material or bone graft substitute.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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